Abstract

It is generally accepted that the primary pharmacological activities and adverse effects of Ephedra Herb are caused by ephedrine alkaloids. Interestingly, our research shows that Ephedra Herb also has ephedrine alkaloid-independent pharmacological actions, such as c-MET inhibitory activity. This study describes the preparation of an ephedrine alkaloids-free Ephedra Herb extract (EFE) by ion-exchange column chromatography, as well as in vitro and in vivo evaluation of its pharmacological actions and toxicity. We confirmed that EFE suppressed hepatocyte growth factor (HGF)-induced cancer cell motility by preventing both HGF-induced phosphorylation of c-Met and its tyrosine kinase activity. We also investigated the analgesic effect of EFE. Although the analgesic effect of Ephedra Herb has traditionally been attributed to pseudoephedrine, oral administration of EFE reduced formalin-induced pain in a dose-dependent manner in mice. Furthermore, we confirmed the anti-influenza virus activity of EFE by showing inhibition of MDCK cell infection in a concentration-dependent manner. All assessments of toxicity, even after repeated oral administration, suggest that EFE would be a safer alternative to Ephedra Herb. The findings described here suggest that EFE has c-Met inhibitory action, analgesic effect, and anti-influenza activity, and that it is safer than Ephedra Herb extract itself. Therefore, EFE could be a useful pharmacological agent.

Effects of EFE, Ephedra Herb extract, and c-Met inhibitor SU11274 on HGF-induced motility and viability of MDA-MB-231 cells. a MDA-MB-231 cells (5 × 104 cells) were suspended in DMEM with or without 40 μg/ml Ephedra Herb, 40 μg/ml EFE, or 5 μM SU11274, and poured into the upper well of the trans-well system. The lower well of the trans-well system contained 600 µL of DMEM containing 50 ng/ml HGF. After 20 h, the cells that had migrated into the lower well were counted. Each assay was performed in triplicate. The error bars represent standard deviation. Statistical significance was determined by Dunnett’s test. **p < 0.001 vs. the number of cells that migrated following HGF stimulation. b 5 × 104 cells were suspended in 100 μl of DMEM containing 50 ng/ml HGF with or without 40 μg/ml Ephedra Herb, 40 μg/ml EFE, or 5 μM SU11274. After 20 h, cell viability was analyzed using the Cell Counting Kit-8 as described in the “” section. Viability (%) is expressed as (absorbance of cells in DMEM containing HGF and crude drug extract/absorbance of cells in DMEM containing HGF) × 100. Each assay was performed in triplicate. The error bars represent standard deviation. Statistical significance was determined by Dunnett’s test. c 5 × 104 cells were suspended in DMEM with 0, 10, 20, or 40 μg/ml EFE and poured into the upper well of the trans-well system. The lower well of the trans-well system contained DMEM with 50 ng/ml HGF. After 20 h, the cells that had migrated to the lower well were counted. Each assay was performed in triplicate. The error bars represent standard deviation. Statistical significance was determined by Dunnett’s test. *p < 0.01, or **p < 0.001 vs. the number of cells that migrated without EFE exposure

Effects of EFE, Ephedra Herb extract, and SU11274 on HGF-induced phosphorylation of c-Met, and effects of EFE and Ephedra Herb extract on the tyrosine-kinase activity of c-Met. a MDA-MB-231 cells were incubated in DMEM, DMEM containing 50 ng/ml HGF, or DMEM containing 50 ng/ml HGF with 10 μg/ml EFE, 10 μg/ml Ephedra Herb extract, or 5 μM SU11274 for 15 min at 37 °C. Tyrosine phosphorylation of c-Met was determined by immunoprecipitation and Western blot analysis. b MDA-MB-231 cells were incubated in DMEM containing 50 ng/ml of HGF with 0, 0.5, 1, 5, or 10 μg/ml of EFE for 15 min at 37 °C. The level of tyrosine phosphorylation of c-Met in the cells was determined by immunoprecipitation and Western blot analysis. c The kinase activity of c-Met was measured using the ProfilerPro Kit. A recombinant c-Met kinase domain was pre-incubated with and without a twofold serial dilution of 8 μg/ml EFE or Ephedra Herb extract at 28 °C for 15 min. The fluorescence-labeled peptide substrate, 1.5 μM 5-carboxyfluorescein-EAIYAAPFAKKK-NH2, and 79.5 μM ATP were added, followed by incubation at 28 °C for 90 min. The kinase reactions were terminated by the addition of 3 mM EDTA. Phosphorylated peptides were separated from substrate peptides and quantified using a LabChip 3000

Effects of EFE and Ephedra Herb extract on formalin-induced pain. ICR mice were treated orally with water, 350 mg/kg EFE, 700 mg/kg EFE, or Ephedra Herb extract for 3 days. On the third day of treatment, formalin tests were performed 6 h after drug or placebo administration. The amount of time that each animal spent licking the injection paw was recorded for 30 min in two phases, the first (0–5 min) and second (15–30 min) phases. Statistical significance was determined by Dunnett’s test. *p < 0.05 or **p < 0.01 vs. control

Effects of EFE and Ephedra Herb extract on influenza virus infection in MDCK cells. MDCK cells (3 × 104 cells) were incubated in 100 μl of 10 % FCS-MEM in a 96-well plate for 24 h, and then washed with MEM. They were incubated for 72 h at 37 °C in 100 μl of MEM or MEM containing a twofold serial dilution of 50 μg/ml EFE or 50 μg/ml Ephedra Herb extract with (b) or without (a) 100 TCID50 of influenza virus A/WSN/33(H1N1). Next, living cells were stained with crystal violet and the absorbance (560 nm) of each sample was quantified using a microplate reader